Subtilisin-like proprotein convertases (SPCs) are endoproteases that function within the secretory pathway of eucaryotic cells. SPCs participate in the maturation of a diverse array of physiologically significant proproteins, including blood proteins, hormones and growth factors, receptors, and extracellular matrix proteins. These enzymes also play a role in the development of disease conditions by endoproteolytically activating bacterial toxins, viral glycoproteins, and proteins involved in tumor progression. The goal of this project is to investigate the substrate specificity of four widely expressed SPCs designated furin, PACE4, PCS, and PC7, each of which has unique substrate preferences. Research focuses on two key issues that may explain substrate specificity in these enzymes. First, substrate amino acids within and adjacent to the endoproteolytic cleavage recognition sequence may be critical for allowing a particular substrate to be recognized by a particular SPC. This possibility will be investigated by using site-directed mutagenesis to alter amino acids in substrate proteins that can be endoproteolytically processed by all of these SPCs. The ability of the enzymes to process the mutant substrates will be examined in vivo, using co-expression experiments, and in vitro. Second, key amino acids within the catalytic domain of the SPC may also play a role in substrate selectivity. Site-directed mutagenesis will be utilized to alter these residues, focusing on PACE4. The consequences of these changes for the substrate preference of the enzyme will be assessed through co-expression experiments. Molecular modeling will be used to place experimental outcomes into a structural context. These experiments will help elucidate mechanisms underlying the distinct substrate preferences of the SPCs.